Method of combating enterobacter infections

ABSTRACT

7-MANDELAMIDO OR 7-AMINOPHENYLACETAMIDO CEPHALOSPORINS BEARING A 3-HETEROCYCLICTHIOMETHYL SUBSTITUENT ARE EFFECTIVE IN COMBATING ENTEROBACTER INFECTIONS. FIVEMEMBERED HETEROCYCLIC RINGS HAVING AT LEAST TWO HETERO ATOMS ARE PREFERRED.

United States Patent O1 Patented Mar. 12, 1974 3,796,801 METHOD OF COMBATING ENTEROBACTER INFECTIONS Joseph R. Guarini, Malvern, Pa., assignor to Smithkline Corporation, Philadelphia, Pa.

No Drawing. Filed Nov. 4, 1971, Ser. No. 195,820 Int. Cl. A61k 27/00 US. Cl. 424-246 12 Claims ABSTRACT OF THE DISCLOSURE 7-mandelamido or 7-aminophenylacetamido cephalosporins bearing a 3-heterocycl'icthiomethyl substituent are effective in combating Enterobacter infections. Fivemembered heterocyclic rings having at least two hetero atoms are preferred.

This invention relates to a method of combating Enterobacter infections. In particular, the invention relates to a method of combating Enterobacter infections by administering to an infected or susceptible warm-blooded mammal a cephalosporin compound of Formula I.

where Ar is phenyl or thienyl;

X is OH or NH M is H or an alkali metal cation, and

Het is a S-membered heterocyclic ring having at least two hetero atoms from the group consisting of S, N, and O. The heterocyclic ring may be substituted with one or more lower alkyl (14 carbons) groups or a trifluoromethyl group.

Cephalosporin compounds have been known for a number of years to possess valuable antibacterial activity, both against Gram-positive and Gram-negative bacteria. They have not, however, been particularly effective against Enterobacter infections. Now we have found that a limited group of cephalosporins, defined by Formula I, possesses sufiicient activity against infections caused by organisms of the genus Enterobacter to enable them to be used effectively in the treatment or prevention of such infections.

The preferred compounds are those where Het is 1,3,4- thiadiazol-Z-yl, 1,3,4-oxadiazol-2-yl, 1,2,4-triazol-3-yl, or 1,2,3,4-tetrazol--yl and the C or N-lower alkyl (1 to 4 carbon atoms) or trifluoromethyl derivatives thereof. Particularly preferred are 1-methyl-l,2,3,4-tetrazol-5-y1, S-methyl-1,3,4-thiadiazol-2-yl, 5-methyl-1,3,4-oxadiazol- 2-yl, and 4,5-dimethyl-1,2,4-triazoI-3-yl. Other preferred groups are l-methylimidazol-Z-yl, 4-methylthiazol-2-yl, 4-methyl-1,2,4-triazol-3-yl, S-methyl-1,2,4-triazol-3-yl, 5- ethyl-1,3,4-thiadiazol-2-yl, S-trifiuoromethyl-1,2,4-triazol- 3-yl thiazol-Z-yl, S-methylthiazol-Z-yl, 3-methylisothiazlol- 5-yl, 2-thiazolin-2-yl.

Also included within the scope of the invention are the pharmaceutically acceptable, nontoxic base salts of the compounds. These include alkali and alkaline earth metal salts and organic ammonium salts.

The compounds of the invention are prepared from starting materials which are known, readily prepared by known methods, or described herein.

In particular, they are prepared by a series of reactions, the essential reactions being displacement of the 3- acetoxy group of 7-aminocephalosporanic acid (7-ACA) with the desired heterocyclic thiol compound and acylation of the 7-amino group of 7-ACA with the appropriate glycine or glycolic acid. The order in which these two reactions are carried out is not critical. The displacement reaction can be followed by observing the disappearance of the acetoxy carbonyl band in the infra red absorption spectrum.

The carboxyl group of the glycines and glycolic acids must be activated prior to the acylation of the 7-ACA compound. Methods of activation include conversion to the acid chloride, mixed anhydride, 2,4-dignitrbphenyl ester, or N-hydroxysuccinimide ester, or reaction first with a carbodiimide or a carbonyl diimidazole. These methods are all well known to one skilled in the art of chemistry.

When X is NH the amino group of the amino acid is protected with one of the well-known protecting groups such as t-butoxycarbonyl, carbobenzyloxy or trichloroethoxycarbonyl. When X is OH, the hydroxy group of the glycolic acid is protected with a common protecting group such as dichloroacetyl, trimethylsilyl, formyl, or tetrahydropyranyl. Following the displacement and/or acylation reactions the protecting group is removed by conventional methods. When X is NH and the protecting group has been removed by acid hydrolysis, the zwitterion product may be obtained by several methods. These include utilization of ion-exchange resins or dissolution of the salt in water and adjustment of the pH to the isoelectric point of the compound with a base such as NaHCO NH OH, tributylamine, t-butylamine, or triethylamine.

Purification of the cephalosporin compounds may be effected by conventional chromatographic or recrystallization techniques. In column chromatography, they can be eluted from silica gel columns with 3% or 5% methanol in chloroform or similar solvent systems. When X is OH the cephalosporin may be purified by conversion to its sodium, potassium or similar salt by dissolution of the compound in a suitable solvent, such as methanol or 20% i-propanol in ethyl acetate, and addition of a solution of a salt of a weaker acid, such as sodium methoxide in methanol or potassium 2-ethylhexanoate in isopropanol. The free acid can be regenerated by dissolution of the salt in water, acidifying the solution, and collection of the product.

Due to the asymmetric a-carbon of the 7-acetamido substituent, optical isomers can exist. The D'-isomer is the preferred isomer; however, it is to be understood that the invention includes also the use of the L-isomer and the racemic D,L-mixture.

Compounds where X is OH are administered by injection. Compounds where X is NH are administered orally or by injection. Injectable formulations are prepared by standard methods using the eompounds or its salt in water or saline solution. Oral formulations, such as capsules, are prepared by standard methods. The dose used to combat Enterobacter infections will vary with the severity of the infection and the age, weight and conditions of the subject, but will generally be in the range of l-24 grn./day, preferably 2-4 gm./day. The dosage will be divided into smaller doses given 4 times per day, although, when a higher dosage is required, the total may be divided into smaller doses given every 2 hours.

The compounds of this invention have been tested extensively and exhibit unusual and unexpectedly high antibacterial activity against Enterobacter aerogenes. In the conventional agar-inclusion test, minimum inhibitory concentrations (MICs) were as low as 3.1 to 6.3 rgJ-ml. In particular, 3-(5 methyl 1,3,4 thiadiazol-Z-ylthiomethyl) 7 (D(-) mandelamido) 3 cephem-4- carboxylic acid had a modal MIC of 3.1 g/ml. when tested against 17 strains of Enterobacter. The same compound had a median eflfective dose (ED against Enterobacter of 7.2 rug/kg. when administered subcutaneously to mice 1 and hours post infection. Under the same test conditions 7-mandelamidocephalosporanic acid had an ED of 36 mg./'kg. and 7-(2-tetrazoleacetamido) -3-(5-methyl-1,3,4-thiadiaZol 2 ylthiomethyl)-3- cephem-4-carboxylic acid had an ED of 9.6 mg./'kg.

The compound 3 (5 methyl 1,3,4 thiadiazol 2- ylthiomethyl) 7 mandelamido 3 cephem 4 carboxylic acid, as well as the other compounds of Formula I, possesses activity against a variety of gram-positive and gram-negative bacteria in addition to Enterobacter. Activity against Nez'sseria gonorrhoeaje is high. Activity against S. aureus, S. pyogenes, S. faecwlis, D. pneumoniae, E. coli, K. pneumoniae, Sa'lmonella spp., and Shigella spp. is also found. The subcutaneous ED of this compound against E. coli species was found in different tests to be 3.0, 6.2, 3.6 and 7.5. Its LD i.v. is approximately 3 g./kg. in rats and 2.5 g./=kg. in mice. It is comparatively free from nephrotoxic effects and its sodium salt is less irritating than either cephalothin or cefazolin.

The following examples are intended to illustrate the preparation of compounds of the invention but are not to be construed as limiting the scope thereof. The MIC values are expressed in ,ug/ ml.

EXAMPLE 1 3-( l-methyltetrazo1-5-ylthiomethyl) -7-D mandelamido- 3-cephem-4-carboxylic acid To a suspension of 7-ACA (27.2 g. 0.1 mole) in a water-acetone (2:1) mixture (300 ml.) was added 5% NaI-ICO until solution was complete and pH 7.5 attained. The reaction solution was heated rapidly to 45 and then 5 mercapto 1 methyltetrazole (17.4 g., 0.15 mole) in acetone (200 ml.) was added. The solution was heated to gentle reflux and maintained at pH 7.5 by addition of 6 N HCl. 1.25 Hours after thiol addition the reaction was cooled to and acidified to pH 3.9 with 6 N 'HCl to give a solid which was collected and washed with water and acetone to yield 14 g. of crude 3-(l-methyltetrazol-5- ylthiomethyl)-7-amino3-cephem-4-carboxylic acid.

This product (6.56 g., 0.02 mole) was suspended in 3% NaHCO (150 ml.) and acetone (150 ml.) and was cooled to 15. To this suspension was added dropwise with stirring over a minute period a solution of D- dichloroacetyhnandelyl chloride (5.62 g., 0.02 mole) [British Pat. 962,024 (1964)] in acetone (40 ml.). Stirring was continued at 15 for minutes, at room temperature for one hour, and then the solution was adjusted to pH 9.3 using 5% Na-HCO and stirred for 1.5 hours. The aqueous solution was extracted with ether, layered with ethyl acetate, cooled and the pH adjusted to 1.5. The organic phase was separated and the aqueous phase extracted with ethyl acetate. The combined organic phases were dried, reduced in volume, and petroleum ether added to precipitate the solid title compound.

The product was purified by chromatography on silica gel, using a 95:5:1 chloroform:methanol:fonnic acid system as eluent. Traces of formic acid were removed by dissolving the product in ethyl acetate, washing with water, drying and evaporating the ethyl acetate in vacuo.

The sodium salt was prepared by adding a solution of sodium 2-ethylhexanoate in i-propyl alcohol to the free acid dissolved in 80:20 ethyl acetatez i-propyl alcohol. Solid was collected, washed with ether and dried.

The modal MIC of this compound against seven strains of Enterobacter aerogenes was 3.1.

EXAMPLE 2 3- S-methyl- 1,3 ,4-thiadiazol-2-ylthiomethyl) -7- [D mandelamido]-3-cephem-4-carboxylic acid A solution of 15.0 g. (0.034 mol) 7-(D-()-mandelamido)cephalosporanic acid methanolate, 4.70 g. (0.036 mol) S-methyl-Z-mercapto-1,3,4-thiadiazole, and 2.99 g.

(0.036 mol) sodium bicarbonate in 300 ml. phosphate buffer (pH 6.4) was stirred under nitrogen at 56 for 21 hr. The clear yellow solution was acidified to pH 2 with dil. hydrochloric acid, and the resulting mixture extracted three times with ethyl acetate. The combined extracts were dried (Na SO and concentrated in vacuo, yielding 16 g. crude free acid.

This free acid was chromatographed on 170 g. silica gel (deactivated with 30 g. H 0) and the desired product (9.44 g.) was eluted with 3% methanol in chloroform.

The sodium salt was prepared by adding 2.88 g. (0.020 mol) sodium 2-ethyl hexanoate in acetone-isopropanol to the free acid (9.44 g., 0.020 mol) in ml. acetone. The resulting precipitate was collected and dried in vacuo, yielding 7.4 g. product.

EXAMPLE 3 3-(3,4'dimethy1-1,2,4tniazol-5-ylthiomethyl)-7-(D-2- aminophenylacetamido)-3-cephem-4-carboxylic acid To a solution of NaHCO (4.2 g., 0.05 mol) in water (50 ml.) was added 7-ACA (6.8 g., 0.025 mol), water (50 ml.), and acetone (25 ml.). The resulting solution was warmed to 45 and then a solution of 3,4-dimethyl-5- mercapto-1,2,4-triazole (5.0 g., 0.038 mol) in acetone (50 ml.) and 5% NaHCO (10 ml.) was added. The reaction was refluxed for 2.75 hours during which time the pH was maintained at ca. 7.6. The solution was cooled to 10 and the pH was adjusted to 3.5 with 3 N HCl. The precipitated product was collected, washed with acetone, and dried to give a 60% yield of 3-(3,4-dimethyl-1,2,4-triazol- 5-ylthiomethyl)-7-amino-3-cephem-4-carboxylic acid.

To a cold solution (-10) of D-a-t-butoxycanboxamidophenylacetic acid (2.26 g., 0.013 mol) and triethylamine (1.4 ml., 0.013 mol) in dry THF (50 ml.) was added with stirring isobutyl chloroformate (1.5 ml., 0.013 mol) over a 10 minute period. To this was added dropwise at 10 a cold solution of the above 7-ACA derivative triethylamine salt [prepared by adding triethylamine (1.5 ml., 0.013 mol) to a suspension of 7-ACA compound (5.0 g., 0.013 mol) in 40 ml. 50% aq. THF]. The reaction solution was stirred for 1 hour at 0 and then for 1.5 hours at room temperature. The 'FHF was evaporated, water added to the mixture, and extracted with ethyl acetate. The organic layer was discarded. The aqueous phase was cooled, layered with ethyl acetate, and acidified to pH 3 using 3 N 'HCl. The phases were separated and the aqueous phase was extracted with ethyl acetate. Evaporation of the combined and dried organic layers gave a solid. Trituration with ether-petroleum ether followed by recrystallization from methylene chlorideether gave the pure t-butoxycanbonyl derivative.

The above product (6.5 g.) was added to cold trifluoroacetic acid (65 ml.) and stirred for 1 hour at 0 and then 0.5 hour at room temperature. The solution was poured slowly into a large volume of ether and the precipitated TFA salt was collected.

The TFA salt (5.3 g.) was dissolved in water (25 ml.) and 15 g. of a polystyrene-amine ion-exchange resin (Amberlite IR-45) was added. After stirring for 1 hour at room temperature the resin was filtered off and the aqueous solution was lyophilized to yield the title compound.

An alternative procedure to obtain the zwitterion involved dissolution of the TFA salt in water, addition of methyl isobutyl ketone (MIBK) and while stirring adjustment of the solution to pH 4 using tri-n-butylamine. The solid product was collected, washed with MIBK and ethyl acetate, and dried.

The MIC of this compound against Enterobacter was 12.5.

EXAMPLE 4 When equivalent amounts of the following mercaptoheterocyclic compounds were substituted for S-mercaptol-methyltetrazole in the procedure of Example 1 the appropriate 3 heterocyclicthiomethyl-7-D-mandelamido 3- cephem-4-carboxylic acids were obtained.

3-mercapto-2-methyl-1,2,4-triazole (MIC against Enterobacter: 25

3-mercapto-4,5-dimethyl-1,2,4-triazole (MIC against Enterobacter=6) 3-mercapto-4-methyl-1,2,4-triazole (MIC against Enterobacter=6) 3-mercapto-5-methyl-1,2,4-triazole (MIC against Enterobacten: 13)

2-mercaptothiazole 4-methyl-Z-mercaptothiazole S-methyI-Z-mercaptothiazole (MIC against Enterob acter= 100) 3-methyl-5-mercaptoisothiazole (MIC against Enterobacter=50) 4-methyl-2-mercaptooxazole (MIC against Enterobacter: 100) *5-rne'thyl-2-mercapto-l,3,4-oxadiazole (modal MIC against 7 strains of Enterobacter=6.3)

3-methyl-5-mercapto-1,2,4-thiadiazole (MIC against Enterobacte1 =25 EXAMPLE 5 When an equivalent amount of 3mercapto-1,2,4-triazole was substituted for the mercaptothiadiazole in Example 2, 3-(1,2,4-triazol-3-ylthiomethy1) 7 (D-mandelamido)-3-cephem-4-carboxylic acid was obtained. The modal MIC of this compound against 7 strains of Enterobacter was 12.5-25.

When an equivalent amount of 3-trifiuoromethyl-5-mercapto-1,3,4-thiadiazole was substituted for the mercaptothiadiazole in Example 2, 7-mandelamido-3-(3-trifluor0- methyl-1,3,4-thiadiazol-5-ylthiomethyl)-3-cephem-4 carboxylic acid was obtained. The MIC of this compound against Enterobacter was 12.5.

EXAMPLE 6 In the procedure of Example 3, when equivalent amounts of the following mercaptoheterocyclic compounds were substituted for the mercaptotriazole, the appropriate 3-(heterocyclicthiomethyl) 7-(D-2 aminophenylacetamido)-3-cephem-4-carboxylic acids were produced.

Z-mercaptothiazole (MIC against Enterobacter: 100) 4-methyl-2-mercaptothiazole (MIC against Enterobacter 100) S-methyl-Z-mercaptothiazole (MIC against Enterobacter: 100

2-mercapto-2-thiazoline S-mercaptoisothiazole (MIC against Enterobacter=100) 5-mercapto-3-methylisothiazole (MIC against Enterobacter: 100) 4-methyl-2-mercaptooxazole (MIC against Enterobacter=200) 3-mercapto-1,2,4-triazole (MIC against Enterob acter: 100) 4-methyl-3-mercapto-l,2,4-triazole (MIC against Enterobacter=25 4-methyl-5-trifluoromethyl-3-mercapto-1,2,4-triazole (MIC against Enterobacter: 25)

S-methyl-B-mercapto-1,2,4-triazole (MIC against Enterobacter=25) 2-mercapto-1,3,4-oxadiazole (MIC against Enterobacter=25 5-methyl-2-mercapto-1,3,4-oxadiazole (modal MIC against 7 strains of Enterobacter was 50) 2-mercapto-1,3,4-thiadiazole (MIC against Enterobacter: 25

5-ethy1-2-mercapto-1,3,4-thiadiazole (MIC against Enterobacter: 25

1-methyl-5-mercaptotetrazole (modal MIC against 7 strains of Enterobacter was 6.3)

S-mercaptotetrazole (MIC against Enterobacter=200) 6 EXAMPLE 7 7-(D-a-aminophenylacetamido)-3-(5-methyl-1,3,4 thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylic acid A solution of 7(D-oz-t butoxycarboxamidophenylacet- -amido)cephalosporanic acid (1.82 g., 3.59 mmol.) [1. L. Spenser et al., J. Med. Chem, 9, 746 1966)] and 5-mercapto-2-methyl-1,3,4-thiadiazole (0.50 g, 3.78 mmole) [1. Sandrtim and I. Wennerbeck, Acta Chem. Scand. 20, 57 (1966)] in 33 ml. of phosphate buffer (pH 6.2) containing bicarbonate (0.315 g., 3.78 mmol.) was heated at 57 for 23 hours. The cloudy solution was cooled to room temperature and extracted with ethyl acetaate. The aqueous phase was cooled in ice, layered with ethyl acetate and acidified to pH 2.0 with 3N hydrochloric acid. The aqueous phase was extracted again with ethyl acetate and the combined, dried (MgSO organic layers were evaporated in vacuo to give 0.79 g. (38%) of desired product as the free acid of the t-butoxycarboxamidophenylacetamidocephalosporin. A portion of the crude product (0.50 g.) was reprecipitated by dissolving it in 5% aqueous sodium bicarbonate solution followed by acidification to give the free acid (0.22 g.) as an off-white solid: M.P. (dec.).

The sodium salt was prepared by treating a solution of the acid in isopropyl alcohol with a solution containing a slight excess of sodium Z-ethylhexanoate in isopropyl alcohol.

The t-butoxycarbonyl derivative (0.22 g.), prepared as described above, was disolved in 3 ml. of cold trifluoroacetic acid and the solution was stirred for 30 minutes at room temperature. The excess trifiuoroacetic acid was evaporated in vacuo at room temperature and the glassy residue was triturated with ether to give 77 mg. of the trifluoroacetate salt of the title compound: IR (Nujol) 5.65 1. (fl-lactam).

The trifluoroacetate salt (77 mg.) was converted to the zwitterionic form by dissolving the salt in 3 ml. of water and stirring the solution at room temperature with 3 ml. of a 25% solution of Amberlite LA-l (acetate form) in methyl isobutyl ketone for 2 hours. The layers were separated and the aqueous phase was evaporated at 30 in vacuo to give 47 mg. of desired product. IR (Nujol) 565a (B-lactam).

The modal MIC of this compound against eight strains of Enterobacter was 12.5.

EXAMPLE 8 7 (D a-hydroxy-2-thienylacetamido)-3-(5-methyl-1,3,4-

thiadiazol 2 ylthiomethyl)-3-cephem-4-carboxylic acid A solution of D-2-thiopheneglycolic acid (2.3 g., 0.015 mol), trimethylsilylacetamide (3.0 g., 0.023 mol) and triethylamine (2.1 ml.) in dry tetrahydrofuran (25 ml.) was refluxed 2 hours in a nitrogen atmosphere. The reaction solution was then cooled to 10, stirred and isobutyl chloroformate (2.5 ml.) was added dropwise; stirring was continued for 20 minutes. To this solution was added dropwise with cooling over a 30 minute period a solution of 7-amino-3-(S-methyl-1,3,4-thiadiazol-5-ylthiomethyl)-3-cephern-4-carboxylic acid (5.16 g. 0.015 mol) (prepared via a procedure similar to that in paragraph one of Example 1) and triethylamine (3 ml.) in 50% aqueous tetrahydrofuran (60 ml.). The reaction was stirred at 5 for 1 hour and room temperature for 1.5 hours. The tetrahydroturan was removed in vacuo, water (50 ml.) was added and the aqueous solution was extracted with ethyl acetate. This organic phase was discarded. The solution was adjusted to pH 2 and the starting 7-ACA derivative precipitated and was collected (3.3 g.). The aqueous solution was extracted with ethyl acetate and the extracts were dried and evaporated to yield a semisolid product (2.5 g.). The product was dissolved in isopropyl alcohol and treated with cyclohexylamine followed by a large volume of ether. The precipitated salt was collected and recrystallized from chloroform-ether.

The MICs of this compound against Enterobacter were 12.5 and 25.

EXAMPLE 9 7-mandelamido-3-( l-methylimidazol-2-ylthiomethyl)- 3-cephem-4-carboxylic acid A solution of 7.62 g. of 7-mandelamidocephalosporanic acid methanolate, 2.94 g. of NaHCO and 3.0 g. of 2- mercapto-l-methylimidazole in a mixture of 50 ml. of water and 25 ml. of acetone was maintained at reflux for 90 min. It was diluted with 75 ml. of water and the pH adjusted to 3.0. The precipitate produced was filtered and washed with water to give 1.72 g. of product after drying.

The MIC of this compound against Enterobacter was 50.

EXAMPLE 10 7- 2-aminophenylacetamido -3-( l-butyltetrazol-S- ylthiomethyl) -3-cephem-4-carboxylic acid 7 amino-3-(1-butyltetrazol-5-ylthiomethyl)-3-cephem- 4-carboxylic acid was prepared in 70% yield according to the procedure of Example 3, except that the reaction tem perature was 72 and the time 1 /2 hours.

To a stirred suspension of 0.04 moles of this 7-amino compound in 80 ml. of CH C1 cooled to there was slowly added 10.9 ml. (0.08 moles) of triethylamine. There was soon added a solution of 16.7 g. (0.04 moles) of D N-t-butoxycarbonylphenylglycine 2,4-dinitrophenyl ester in 20 ml. CH 'Cl After stirring at 25 overnight, the CH CI was removed in vacuo (20), and 500 ml. of ethyl acetate was added to the residue. The resulting solution was washed with 100 ml. 1 N HCl, the organic layer separated and dried, and the solvent removed to give 7 N t butoxycarbonylaminophenylacetamido 3 (l-butyltetrazol-5-ylthiomethyl)-3-cephem-4- carboxylic acid.

This acid was converted to the free amino product by reaction with cold trifiuoroacetic acid 1:10 w./v.) for 20 minutes. Excess trifiuoroacetic acid was removed in the cold, and the residue triturated with ether to give the trifluoroacetate salt as a white solid. This salt was dissolved in a small amount of water to which a drop of concentrated HCl had been added, and any insoluble material filtered off. The filtrate was basified to pH 3.9 and the product collected by filtration.

This compound had an MIC of 200 against Enterobacter.

EXAMPLE ll 7 -mandelamido-3-(4-methyl-3-trifluoromethyl-1,2,4-triazol-5-ylthiomethyl)-3-cephem-4-ca.rboxylic acid To a cooled solution of O-tetrahydropyranylmandelic acid (4.73 g., 0.02 mol) and N-hydroxysuccinimide (2.3 g., 0.02 mol) in anhydrous THF was added dicyclohexylcarbodiimide (4.12 g., 0.02 mol). After stirring for 7 hours at ice bath temperature, the reaction was allowed to stand at room temperature overnight. The dicyclohexylurea was removed by filtration and the filtrate was evaporated to yield the succinimide ester.

7 amino 3 (4-methyl-3-trifluoromethyl-l,2,4-triazol- 5-ylthiomethyl)-3-cephem-4-carboxylic acid was prepared according to the procedure of Example 3 by substituting 5 mercapto 4 methyl-3-trifluoromethyl-1,2,4-triazole for 3,4-dimethyl-5-mercapto-1,2,4-triazole.

To a cooled solution of this 7-aminocephem compound (6.3 g., 0.016 mol) in 80 ml. of dry pyridine containing 3.4 g. triethylamine was added the succinimide ester (5.5 g., 0.016 mol). The reaction was stirred 3 hours at room temperature and then poured into 600 ml. water that had been acidified to pH 2. The precipitated solid An injectable composition is formed by adding 2 ml. sterile water or sterile isotonic saline to 500 mg. of the cephalosporin sodium salt.

EXAMPLE 13 An antibacterial capsule has the following components: Mg. Cephalosporin 500 Lactose 2%) Magnesium steara-te What is claimed is:

1. A method for combatting Enterobacter infections comprising administering to a warm-blooded mammal infected with an Enterobacter infection an effective amount for treating said Enterobacter infection of a composition comprising a cephalosporin of the formula:

s mono 0 NH- X on s Het (i1 0 OM where Ar is phenyl or thienyl; X is OH or NH M is H or an alkali metal or ammonium cation; and Het is 1,2,4, triazole, unsubstituted or substituted with a lower alkyl or trifiuoromethyl group at one or two carbon or secondary nitrogen atoms, and a suitable pharmaceutical carrier. 2. A method as set forth in claim 1 where Ar is phenyl, M is H, and X is OH.

3. A method as set forth in claim 2 where Het is 4- methyl-S-trifiuoromethyl- 1 ,2,4-triazol-3 -yl.

4. A method as set forth in claim 2 where Het is 4- methyl-l,2,4-triazol-3-yl.

5. A method as set forth in claim 2 where Het is 5- methyl-1,2,4-triazol-3-yl.

6. A method as set forth in claim 2 where Het is 4,5- dimethyl-l ,2,4-triaZol-3 -yl.

7. A method as set forth in claim 2 where Het is 2- methyl-1,2,4-triazol-3-yl.

8. A method as set forth in claim 1 where Ar is phenyl, M is H, and X is NH 9. A method as set forth in claim 11 where Het is 3,4- dimethyl-1,2,4-triazol-5yl.

10. A method as set forth in claim 8 where Het is 3- methyl-1,2,4-triazol-6-yl.

11. A method as set forth in claim 8 where Het is 4- methyl-1,2,4-triazol-3-yl.

12. A method as set forth in claim 1 where Ar is thienyl.

References Cited UNITED STATES PATENTS 3,641,021 2/1972 Ryan 260-243 C JEROME D. GOLDBERG, Primary Examiner 

